Kaohsiung J Med Sci June 2010 • Vol 26 • No 6 290
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destructive arthropathy. In addition to joints, internal organs may also be in-volved. Genetic and environmental factors may be involved in the pathogenesis of RA [1–3]. Although the detailed pathogenesis is still unclear, cytokines may play an important role in the perpetuation of arthritis
[4]. A disordered balance between pro- and anti-inflammatory cytokine production can be found in rheumatoid synovitis. The production of pro- and anti-inflammatory cytokines in the synovial membrane is increased, although the balance is shifted toward pro-inflammatory cytokines.
The suppressor of cytokine signaling (SOCS) and cytokine-inducible SH2 (CIS) protein are a family of cytokine-inducible negative regulators of cytokine sig-naling [5]. There are eight members in the CIS/SOCS family of proteins including CIS, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7. They have a central SH2 domain, an N-terminal domain of vari-able lengths and sequences, and a SOCS box in the Received: Oct 27, 2009 Accepted: Jan 22, 2010
Address correspondence and reprint requests to: Dr Jeng-Hsien Yen, Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 Zihyou 1st
Road, Kaohsiung 807, Taiwan. E-mail: [email protected]
I
NCREASED
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XPRESSION OF
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UPPRESSOR OF
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IGNALING
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RNA
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ATIENTS
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HEUMATOID
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RTHRITIS
Hua-Chen Chan,1Liang-Yin Ke,2Ching-Ching Liu,1Lin-Li Chang,3Wen-Chan Tsai,4 Hong-Wen Liu,4and Jeng-Hsien Yen1,4,5
1Graduate Institute of Medicine, 3Department of Microbiology, School of Medicine, College of Medicine, 5Center of Excellence for Environmental Medicine, Kaohsiung Medical University; 2Department of
Laboratory Medicine, and 4Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
The objective of this study was to investigate the associations between suppressor of cytokine signaling 1 (SOCS1) mRNA expression and SOCS1 polymorphisms with the development of rheumatoid arthritis (RA). One hundred and eighty-one patients with RA and 96 healthy controls were enrolled in this study. The SOCS1 mRNA level in peripheral blood mononuclear cells (PBMCs) was detected by quantitative real-time polymerase chain reaction. SOCS1 phisms were determined by the polymerase chain reaction/restriction fragment length polymor-phism method. We found that the expression of SOCS1 mRNA in PBMCs was significantly greater in patients with RA than in healthy controls. There were no significant differences in the expression of SOCS1 mRNA among patients with different disease activities. The increment in SOCS1 mRNA after stimulation with various cytokines was slightly lower in the patients with RA than in the healthy controls. This study also demonstrated that the SOCS1 polymorphisms were not associated with susceptibility to RA. In conclusion, the expression of SOCS1 mRNA in PBMCs is higher in patients with RA than in healthy controls. The increment in SOCS1 mRNA expression in PBMCs after stimulation with different cytokines seems to be lower in patients with RA than in healthy controls.
Key Words:polymorphisms, rheumatoid arthritis, SOCS1 (Kaohsiung J Med Sci 2010;26:290–8)
C-terminal domain. The SOCS box is involved in the recruitment of the ubiquitin-transferase system and mediates protein degradation. SOCS1 and SOCS3 also have a kinase inhibitory region in the N-terminal domain, which inhibits Janus kinase (JAK) tyrosine kinase activity. Therefore, SOCS1 and SOCS3 seem to have a combined effect on kinase inhibition by kinase inhibitory region and proteosomal degradation by the SOCS box. SOCS1 and SOCS3 bind to and inhibit the catalytic activity of JAKs. Then, the complexes may be degraded by ubiquitination and proteasomal degra-dation mediated via the SOCS box. SOCS1 regulates the response of immune cells to cytokines, and regu-lates cytokine-mediated homeostasis, including innate and adaptive immunity [6]. Many cytokines activate JAK, resulting in the activation of signal transducer and activator of transcription (STAT), and subsequent nuclear localization with the induction of gene ex-pression [7]. The binding of STAT to DNA induces the SOCS1 expression and the production of SOCS1, which provides negative feedback to regulate the cytokine–JAK–STAT pathway [6,8]. Meanwhile, SOCS1 may also be induced by nuclear factor kappa B (NF-κB) and, in turn, suppresses NF-κB expression via negative feedback [9,10].
SOCS1 influences the signaling of many cytokines related to inflammatory diseases. The negative regula-tion of cytokine signaling may be impaired if the pro-tein expression of SOCS1 is low. Therefore, SOCS1 may be associated with the development of inflammatory diseases. Isomaki et al reported elevated expression of SOCS1 in peripheral blood mononuclear cells (PBMCs) from patients with RA [11]. However, the study by Tsao et al showed that mRNA expression of SOCS1 in PBMCs was not significantly different between RA patients and healthy controls [12]. Until now, an associ-ation between SOCS1 polymorphisms and RA has not been reported. In this study, we investigated the associ-ations between SOCS1 mRNA expression and poly-morphisms in patients with RA.
M
ETHODSOne hundred and eighty-one patients with RA (147 females and 34 males; mean age± standard deviation, 48.3± 12.6 years) and 96 healthy controls (79 females and 17 males; 45.6± 11.8 years) were enrolled in this study. All of the patients and controls were Taiwanese.
This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital. The diagnosis of RA was made according to the 1987 American College of Rheumatology revised criteria for the classification of RA.
Stimulation of PBMC with various
cytokines
PBMCs were isolated from 10 patients with RA and 10 healthy controls. PBMCs were cultured at 1× 106 cells/mL in complete RPMI medium, and stimulated with 10 ng/mL of recombinant interferon (IFN)-γ, in-terleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNFα) for 1 hour or 16 hours. Then, total RNA ex-traction and quantitative real-time polymerase chain reaction (PCR) were performed to determine SOCS1 mRNA expression.
Total RNA extraction, reverse transcription,
and genomic DNA extraction
The PBMCs were separated by the Ficoll-Paque me-thod. Total RNA was extracted by a NucleoSpin RNA II kit (Macherey-Nagel, GmbH, Düren, Germany). Reverse transcription was performed to obtain cDNA using a high capacity cDNA Archive kit with random primers (Applied Biosystems, City, State, Country). Genomic DNA was extracted from the buffy coat of the peripheral blood using a commercial kit (Geneaid Biotech Ltd.).
Quantitative real-time PCR
For quantitative analysis of SOCS1 mRNA in PBMC, quantitative real-time PCR was performed with the ABI Prism 7000 Detection System (Applied Biosystems, Foster City, CA, USA). In this study, RNA polymerase II (RP II) was used as the endogenous control because of its stable expression in different tissues [13]. The sequences of the primers and TaqMan probes (Applied Biosystems, Foster City, CA, USA) for SOCS1 and RP II mRNA are summarized in Table 1. The real-time PCR condition consisted of an initial incubation at 50°C for 2 minutes, enzyme activation at 95°C for 10 minutes, and 40 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 62°C for 1 minute. The hybridization temperature of the TaqMan probe was 62°C. All of the samples were tested in triplicate. SOCS1 mRNA expression was normalized to RP II and its relative expression was calculated using the 2−⌬CTmethod. A validation test was also performed
to determine the PCR efficiency of the target and endogenous controls. The results showed that the amplification efficiencies of SOCS1 and RP II were approximately equal.
Polymorphisms of SOCS1
There are several polymorphisms in the promoter region of SOCS1 and one non-synonymous polymor-phism (rs 11549428; 1335 C/G, amino acid 210, His Gln) in exon 2 of SOCS1. Harada et al showed that the
SOCS1 promoter polymorphism –1478 del enhanced
the transcriptional level of SOCS1 [14]. The biallelic mutation in the SOCS box, which is encoded by exon 2 in SOCS1, might result in impaired JAK2 degrada-tion and sustained JAK2 activadegrada-tion [15]. Therefore, the polymorphisms –1478 CA/del and 1335 C/G were investigated in this study. A polymorphism 1351 C/A (rs 1801729; exon 2) in the 3’-untranslated region was also determined. These polymorphisms were deter-mined by the PCR/restriction fragment length poly-morphism method. The sequences of primers and restriction enzymes are summarized in Table 2. To determine the SOCS1 –1478 CA/del polymorphisms, PCR was done under the following conditions: initial denaturation at 95°C for 5 minutes, and 30 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute and extension at 72°C for 1 minute, with a final extension at 72°C for 7 minutes. The PCR product was digested with DdlI.
The amplification conditions for the determina-tion of the SOCS1 1335 G/C and 1351 C/A polymor-phisms were as follows: initial denaturation at 96°C for 3 minutes, followed by five cycles of denaturation at 96°C for 1 minute; annealing at 60°C for 1 minute and extension at 72°C for 1 minute; and 25 cycles of denaturation at 96°C for 1 minute; annealing at 55°C
for 1 minute and extension at 72°C for 1 minute; and a final extension at 72°C for 7 minutes. The restriction enzymes, Hpy188III and HhaI, were used to deter-mine the SOCS1 1335 G/C and SOCS1 1351 C/A polymorphisms, respectively.
Serum cytokine concentrations
The serum concentrations of TNFα and IFNγ were determined using commercially available enzyme-linked immunosorbent assays (eBioscience Inc., San Diego, CA, USA). All of the samples were tested in duplicate.
Clinical manifestations
The disease activity score 28 (DAS28) was used to assess the disease activities of RA patients [16,17]. The disease activities of RA were defined as low activity (3.2≥ DAS28 > 2.6); moderate activity (5.1 ≥ DAS28 > 3.2); and high activity (DAS28 > 5.1). The Sharp score was also used to assess joint involve-ment [18].
Statistical analysis
Student’s t test was used to compare SOCS1 mRNA expression between the RA patients and the controls. SOCS1 mRNA expression was compared among the patients with RA according to disease activity using one-way analysis of variance. The Mann-Whitney U test was used to compare the effects of cytokine stim-ulation on PBMC SOCS1 mRNA between the patients with RA and the controls. The χ2 test was used to compare the genotype distributions of SOCS1 poly-morphisms between the patients with RA and the controls. Pearson’s correlation test was used to evalu-ate the correlation between SOCS1 mRNA level and Sharp score.
Table 1.Sequences of the primers and probes to detect suppressor of cytokine signaling 1 and RNA polymerase II in
quantitative real-time polymerase chain reaction
Gene Primers/probes Sequences
SOCS1 forward 5’-GGG AGC GGA TGG GTG TAG G-3’ reverse 5’-AGA GGT AGG AGG TGC GAG TTC-3’
probe 5’-FAM-CGC GAT CGG GAC CCA GAG GGA GCA CCA GGA GAT CGC G-TAMRA-3’
RNA Polymerase II forward 5’-GCA CCA CGT CCA ATG ACA T-3’ reverse 5’-GTG CGG CTG CTT CCA TAA-3’
probe 5’-HEX-TAC CAC GTC ATC TCC TTT GAT GGC TCC TA-BHQ-3’ SOCS1= Suppressor of cytokine signaling 1.
R
ESULTSThis study demonstrated that the expression of SOCS1 mRNA was significantly higher in the PBMCs of patients with RA than in the controls (Figure 1;
p < 0.001).
Table 3 shows the SOCS1 mRNA expression in pa-tients with RA according to disease activity. One-way analysis of variance revealed no differences in SOCS1 mRNA levels according to disease activity.
Figure 2 shows the SOCS1 mRNA expression in PBMCs stimulated with various cytokines (10 ng/mL) for 1 or 16 hours, from 10 patients with RA and 10 healthy controls. In the healthy controls, the expres-sion of SOCS1 mRNA increased in response to stimu-lation with the cytokines, particularly with IFNγ, for 1 hour. In contrast, similar findings were not found in PBMCs from patients with RA except in response to stimulation with IFNγ. In PBMCs from patients with RA, there were no marked changes in the SOCS1 mRNA levels after stimulation with IL-1β, IL-6, and TNFα. The increments in SOCS1 expression in the RA patients were lower than those of the controls. The expression of SOCS1 mRNA, after stimulation with IFNγ for 16 hours was significantly lower in PBMCs from patients with RA than in those from healthy controls (p< 0.05). A similar finding was also found after stimulation with IL-1β (p = 0.008).
The SOCS1 1335G and 1351A alleles were not de-tected in this population of Taiwanese people (Table 4). There were no significant differences in the genotype frequencies of SOCS1 –1478 CA/del polymorphisms between the patients with RA and the healthy controls.
D
ISCUSSIONThis study showed that the expression of SOCS1 mRNA in PBMCs was significantly higher in patients with RA than in healthy controls. However, the increment
Table 2.Sequences of primers and restriction enzymes for detecting suppressor of cytokine signaling 1 polymorphisms
Polymorphisms Primers Restriction enzymes −1478 CA/del 5’-TGTCGTCCAGCTGCACCTC-3’ Dde I
5’-ACC ACA GGC TTC AGA GGA AC-3’
1335 G/C & 1351 C/A 5’-GCT GCT GGA GCA CTA CGT G-3’ Hpy188III (for 1335 G/C) 5’-CCAGATACAGTTAAGCTGCTAC-3’ HhaI (for 1351 C/A)
Figure 1.Suppressor of cytokine signaling 1 mRNA expression
normalized for RNA polymerase II in peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis and in healthy controls. RA=Rheumatoid arthritis; SOCS1 = suppressor of cytokine signaling 1; RP II=RNA polymerase II; SD =standard deviation. (Control vs. RA 95% confidence interval=−0.77 to −0.3; p< 0.001). Control 12 10 8 6 4 2 0 RA SOCS1/RP II 12 10 8 6 4 2 0 SOCS1/RP II Mean± SD = 0.20 ± 0.47 Mean ± SD = 0.73 ± 1.41
in SOCS1 mRNA expression in response to stimula-tion with IFNγ and IL-1β were significantly lower in PBMCs obtained from patients with RA than in those from healthy controls.
Many cytokines, including IL-2, IL-4, IL-6, IL-9, IL-13, IFNα, IFNβ, IFNγ and TNFα, may induce SOCS1
Table 3. Suppressor of cytokine signaling 1 mRNA
expression in peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis according to disease activity*
SOCS1 mRNA expression SOCS1/RP II
High 0.65± 0.86
Moderate 0.90± 1.77
Low 0.74± 1.47
*Data presented as mean± standard deviation. There were no significant differences in the levels of SOCS1 expression among the patients with different disease activities. RP II=RNA poly-merase II; SOCS1= suppressor of cytokine signaling 1.
expression [19]. Colony stimulatory factors, hormones and growth factors may also induce the expression of SOCS1. Furthermore, SOCS1 expression is induced in macrophages by lipopolysaccharide (LPS) stimulation [9]. In turn, SOCS1 inhibits LPS-induced NF-κB and STAT1 activation [9]. Similarly, Wesemann et al showed that SOCS1 inhibited IFNγ-induced TNFα secretion and subsequent NF-κB activation [20]. These effects of SOCS1 may be mediated by SOCS1-induced degrada-tion of the p65/NF-κB subunit [21]. Therefore, SOCS1 is induced by STAT and NF-κB, and suppresses both STAT1 and NF-κB.
SOCS1 is involved in innate immunity and plays a significant role in the pathogenesis of inflammatory dis-eases [22]. SOCS1 deficient mice developed multi-organ failure associated with severe inflammation [23,24]. Moreover, joint inflammation and destruction were significantly enhanced in SOCS1-deficient mice. An
0.4500 0.4000 0.3500 0.3000 0.2500 0.2000 0.1500 0.1000 0.0500 0.0000 SOCS1 mRNA expr ession
RPMI IFN-γ IL-1β IL-6 TNFα
∗ † 0.8000 0.7000 0.6000 0.5000 0.4000 0.3000 0.2000 0.1000 0.0000 SOCS1 mRNA expr ession
RPMI IFN-γ IL-1β IL-6 TNFα
A B
Controls RA Controls
RA
Figure 2.Suppressor of cytokine signaling 1 mRNA expression in PBMCs from 10 patients with rheumatoid arthritis and 10 healthy
controls in response to stimulation with various cytokines (10 ng/mL) for (A) 1 hour or (B) 16 hours. The Mann-Whitney U test was used for statistical analysis. *p< 0.05 (Control vs. patients with rheumatoid arthritis); †p= 0.008 (Control vs. patients with rheumatoid
arthritis). RPMI= negative control; IFN = interferon; IL = interleukin; SOCS1 = suppressor of cytokine signaling 1; TNF = tumor necrosis factor; RA= patients with rheumatoid arthritis.
Table 4.Frequencies of suppressor of cytokine
signal-ing 1 polymorphisms in the patients with rheumatoid arthritis and healthy controls
SOCS1 RA (n= 181) Healthy control polymorphisms (n= 96) −1478 CA/CA 130 (71.8) 73 (76.9) −1478 CA/del 46 (25.4) 21 (21.8) −1478 del/del 5 (2.8) 2 (2.1) 1335 C/C 181 (100) 96 (100) 1351 C/C 181 (100) 96 (100) RA= Rheumatoid arthritis; SOCS1 = suppressor of cytokine signaling 1.
animal model with deletion of the STAT-binding site in gp130, an IL-6 receptor subunit, resulted in a severe joint disease resembling RA [25,26]. Fujimoto et al reported that SOCS1 expression in mutant mice with restoration of the SOCS1 gene was insufficient for effective downregulation of its target genes [27]. These mice had increased levels of serum immuno-globulins (Ig) and anti-DNA, and glomerulonephritis with glomerular IgG deposition. Therefore, dysfunc-tion of SOCS1 may be a pathologic factor in systemic autoimmune diseases.
Our study demonstrated enhanced expression of SOCS1 in the PBMCs obtained from patients with RA, which might be caused by stimulation by cytokines or other factors. Although SOCS1 mRNA can be upregu-lated by IFNγ, the serum levels of IFNγ were not signif-icantly different between the patients with RA and the controls in this study (RA, 37.00± 34.98 pg/mL; con-trols, 35.00±12.95pg/mL). Thus, the increased expres-sion of SOCS1 in patients with RA may be caused by other factors.
The results of this study are consistent with those reported by Isomaki et al [11], but differ from those reported by Tsao et al [12]. The discrepancy between our study and the study by Tsao et al may be due to different sample sizes or different conditions of the patients with RA.
In this study, the increment in SOCS1 mRNA ex-pression in cultured PBMCs stimulated by various cytokines seemed to be lower in PBMCs obtained from patients with RA than in those from healthy
controls. Furthermore, the SOCS1 mRNA expression after stimulation with IFNγ or IL-1β for 16 hours were significantly lower in PBMCs obtained from patients with RA than in those obtained from healthy controls. Although the mechanisms underlying the lower increment in SOCS1 expression after stimula-tion with cytokines are still unknown, they may be related to preactivation of PBMCs in patients with RA, a state that has been reported in earlier studies [28–30]. The lower increment in SOCS1 mRNA expres-sion may also be related to polymorphisms or DNA hypermethylation in the promoter of SOCS1. However, the genotype frequencies of SOCS1 –1478 CA/del were not significantly different between the patients with RA and the controls in this study. SOCS1 gene silencing caused by hypermethylation of the pro-moter CpG islands has been reported in solid tumors and in hematological malignancies [31–36]. Decreased
SOCS1 gene expression because of DNA methylation
may stimulate liver inflammation [36]. However, it is unknown whether the lower increment in SOCS1 mRNA in patients with RA is related to DNA hyper-methylation of SOCS1.
This study showed that the expression of SOCS1 mRNA was higher in patients with moderate RA dis-ease activity than in those with low disdis-ease activity. In contrast, the SOCS1 mRNA level was slightly lower in patients with high disease activity compared with those with moderate or low disease activity. The rea-sons for these findings are still unknown. Genetic or epigenetic changes in SOCS1 may influence the expres-sion of SOCS1 mRNA. Although there were no differ-ences in the genotype distributions of the SOCS1 –1478 CA/del polymorphisms according to disease activity, it cannot be excluded that other polymorphisms may be related to the different expressions of SOCS1 mRNA. Moreover, other members of the SOCS family may contribute to inflammation in RA.
We found that the SOCS1 mRNA levels were not associated with the Sharp scores of the RA patients (data not shown). Similarly, the SOCS1 –1478 CA/del polymorphisms were not related to the Sharp scores. The reasons are still unknown. Sharp scores are re-lated to the severity of inflammation and disease duration. However, the SOCS1 mRNA levels cannot directly reflect the inflammation status of disease. In our previous study, we found increased expression of SOCS1 mRNA in patients with systemic lupus erythematosus [37].
As a negative regulator of cytokines, SOCS1 might be used in the treatment of cytokine dysregulated disorders [8,38]. Indeed, a tyrosine kinase inhibitor peptide, acting as a mimetic of SOCS1, inhibited IFNγ signaling and suppressed the proliferation of prostate cancer cell lines [39]. Shouda et al also showed that adenovirus-mediated overexpression of SOCS3 pre-vented mouse antigen-induced arthritis or collagen-induced arthritis [40]. In a mouse model of arthritis, SOCS1 can counteract the onset and progression of autoimmune arthritis [41]. SOCS1 was also reported to negatively regulate acute inflammatory arthritis and CD4+T cell activation [41,42]. Therefore, upregulating SOCS1 expression may offer a new therapeutic strat-egy for the treatment of RA [43,44].
In summary, this study showed that there were no significant differences in the genotype frequencies of
SOCS1 polymorphisms between patients with RA and
the healthy controls. The expression of SOCS1 mRNA in PBMCs was significantly increased in those from patients with RA in comparison with those from healthy controls. However, the increment in SOCS1 mRNA in cultured PBMC after stimulation with cytokines was lower in PBMCs from patients with RA than in those from healthy controls.
R
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收文日期： 98 年 10 月 27 日 接受刊載： 99 年 1 月 22 日 通訊作者：顏正賢醫師 高雄醫學大學附設醫院過敏免疫風濕科 高雄市自由一路 100 號
類風濕性關節炎病人之 SOCS1 mRNA 表現增加
詹華蓁1 柯良胤2 劉靜菁1 張玲麗3 蔡文展4 劉宏文4 顏正賢1,4,5 高雄醫學大學 1醫學研究所 3醫學系 微生物學科 5環境醫學頂尖研究中心 高雄醫學大學附設醫院 2檢驗醫學部 4過敏免疫風濕科本研究的主要目的是在探討 suppressor of cytokine signaling（SOCS1）mRNA 含量以及 SOCS1 基因多形性和類風濕性關節炎（Rheumatoid Arthritis, RA）的關 係。本研究共包括 181 位 RA 病人和 96 位正常健康者。周邊血液單核球中 SOCS1
mRNA 含 量 之 測 定 是 用 即 時 定 量 聚 合 酶 連 鎖 反 應（real-time PCR） 方 法， 而 SOCS1 基因多形性則用 PCR/RFLP 方法測定。本研究結果顯示 RA 病人周邊血液
單核球中之 SOCS1 mRNA 含量比正常人顯著增高。不同疾病活動性之 RA 病人間， 其 SOCS1 mRNA 含量並無顯著差異。RA 病人之周邊血液單核球經不同細胞激素刺 激後，其 SOCS1 mRNA 增加的幅度比正常人低。本研究亦顯示 SOCS1 基因多形性 和是否發生 RA 無關。簡言之，RA 病人之周邊血液單核球細胞含有較高的 SOCS1
mRNA。經不同細胞激素刺激後，其增加的幅度似乎比正常人者低。
關鍵詞：基因多形性，類風濕性關節炎，SOCS1 （高雄醫誌 2010;26:290–8）
